The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter

Abstract

Cyanide is traditionally viewed as a cytotoxic agent, with its primary mode of action being the inhibition of mitochondrial Complex IV (cytochrome c oxidase). However, recent studies demonstrate that the effect of cyanide on Complex IV in various mammalian cells is biphasic: in lower concentrations (nanomolar to low micromolar) cyanide stimulates Complex IV activity, increases ATP production and accelerates cell proliferation, while at higher concentrations (high micromolar to low millimolar) it produces the previously known ('classic') toxic effects. The first part of the article describes the cytotoxic actions of cyanide in the context of environmental toxicology, and highlights pathophysiological conditions (e.g., cystic fibrosis with Pseudomonas colonization) where bacterially produced cyanide exerts deleterious effects to the host. The second part of the article summarizes the mammalian sources of cyanide production and overviews the emerging concept that mammalian cells may produce cyanide, in low concentrations, to serve biological regulatory roles. Cyanide fulfills many of the general criteria as a 'classical' mammalian gasotransmitter and shares some common features with the current members of this class: nitric oxide, carbon monoxide, and hydrogen sulfide.

Keywords: bioenergetics; carbon monoxide; hydrogen sulfide; metabolism; mitochondria; nitric oxide.

Fig. 1

Cyanide as a Complex IV inhibitor and mitochondrial poison. CCOx dimer bounded to CN^‐. CCOx is localized in the mitochondrial inner membrane with the cytochrome c binding site exposed to the intermembrane space. The redox centers, namely, Cu_A, heme a, and the binuclear center heme a₃ /Cu_B of a single monomeric unit are represented in different colors. In the reduced state, Cu_B is coordinated by His290, His291, His240, and Y244, and upon binding of cyanide, His290 is displaced, thus allowing the accommodation of cyanide between Cu_B and heme a₃ . From the Protein Data Bank coordinates of the fully reduced bovine heart CCOx in the presence and absence of cyanide (PDB Id: 3AG1 and 3AG4), deposited by Muramoto and colleagues [30, 36].

Fig. 2

Pathways contributing to the cytotoxic actions of cyanide in mammalian cells. (A) Typically, at high micromolar concentrations (e.g., 100–300 µm in neurons), cyanide induces apoptotic cell death. Early stages of this process include mobilization of calcium from intra‐ and extracellular pools. This calcium mobilization (possibly, in combination with a partial inhibition of CCOx and mitochondrial dysfunction, coupled with ER dysfunction), stimulates various effectors of apoptotic cell death. For instance, ROS are generated either by cyclooxygenase (which is stimulated by calcium mobilization) or by the mitochondria (as a consequence of CCOx inhibition). ROS and calcium stimulate various apoptotic effectors (e.g., endonucleases, caspases), BNIP3 (BCL2 and adenovirus E1B 19‐kDa‐interacting protein 3) and signaling pathways (e.g., NF‐κB). These processes culminate in apoptotic cell death; these cells are typically eliminated by phagocytes and do not exacerbate local inflammatory responses. (B) Typically, at low millimolar concentrations (e.g., 1–3 mm), cyanide induces necrotic cell death. A central part of this process is a pronounced inhibition of CCOx and mitochondrial dysfunction, also reflected in severe degree of cellular ATP depletion. An additional factor in this process is calcium overload, followed by activation of PKC. Mitochondrially derived ROS (perhaps together with ROS formed by other cellular sources and perhaps also in combination with NO to form peroxynitrite) induces DNA single strand breakage, which is a direct activator of the nuclear enzyme PARP. PKC activation, and PARP activation, further depletes cellular NAD⁺ and ATP levels. Because of the low cellular ATP, cells are unable to maintain the activity of membrane pumps and the membrane potential dissipates and the cell starts to ‘leak’ and release its intracellular content. During full‐fledged necrosis, all cellular content is released as the cell disintegrates. This process can, in turn, lead to additional local or remote inflammation.

Fig. 3

Physiological cyanide‐generating systems in mammals and other organisms. Solid arrows represent enzyme catalyzed reactions, while dashed arrows stand for nonenzymatic degradation. (A) MPO catalyzes the chlorination of glycine into N‐dichloro‐glycine, which is an unstable compound and decomposes to its corresponding nitrile, followed by nonenzymatic release of cyanide and carbon dioxide. (B) Cyanocobalamin reductase (MMACHC) catalyzes the decyanation of CNCbl to yield cob(II)alamin (Cbl) and cyanide. (C) Carboxyl esterase catalyzes cypermethrin (an insecticide of the family of pyrethroids) hydrolysis to its corresponding cyanohydrin. Cyanohydrins are unstable species which give cyanide as product of degradation. (D) Aliphatic nitriles undergo epoxidation catalyzed by the microsomal enzyme CYP2E1, followed by liberation of cyanide by epoxide hydrolyze (EPHX). (E) In plants β‐glucosidase/hydroxynitrile lyase system is believed to be one of the main sources of free cyanide. Cyanogenic glucosides are processed by β‐glucosidase thus producing the correspondent cyanohydrin, which are then converted to aldehyde (or ketone) by hydroxynitrile lyase, with the concomitant elimination of cyanide. (F) Ethylene in plants is considered a hormone involved in many processes. The ethylene synthesis is accomplished by the oxidation of 1‐aminocyclopropane‐1‐carboxylic acid (ACC), by ACC oxidase, into ethylene and cyanoformic acid. The latter spontaneously decomposes in cyanide and carbon dioxide. (G) Cyanide production from glyoxylate has been observed in algae (Chlorella vulgaris), spinach (Spinacia oleracea), corn (Zea mays), and barley leaves. In the presence of hydroxylamine, glyoxylate generates glyoxylate oxime, followed by degradation into cyanide, carbon dioxide, and water. The enzyme catalyzing this reaction has a molecular mass of 40 kDa and has been established to require for its catalytic activity ADP and Mn²⁺. (H) Camalexin is a characteristic alkaloid of Arabidopsis thaliana accumulated upon infection of a variety of pathogens. The final steps of its biosynthesis are controlled by CYP71B15, which catalyzes both the formation of thiazolidine ring of cysteine‐indole‐3‐acetonitrile, with the concomitant release of cyanide, and the subsequent oxidative decarboxylation of dihydrocamalexic acid to camalexin. (I) Cyanide biosynthesis from aromatic amino acids (histidine, tyrosine, and phenylalanine) has been observed in the alga Chlorella vulgaris and in spinach leaves. The reaction has been shown to be catalyzed by amino acid oxidase co‐incubated with Mn²⁺ and horseradish‐peroxidase, thus causing the formation of cyanide. Several bacterial species (the most extensively characterized being Pseudomonas aeruginosa) are known to produce cyanide. The immediate precursor of cyanide is glycine which is converted to cyanide and carbon dioxide in a reaction catalyzed by hydrogen cyanide synthase.

Fig. 4

Reactions of cyanide in mammalian cells. CN^‐ is a strong‐field ligand as well as a strong nucleophile. As such, it is involved in many reactions in mammalian cells. Metal binding: Cyanide is reactive toward transition metals, including iron, zinc, copper, and cobalt, thus binding many metalloproteins (often with an inhibitory effect) and the corrinoid ring of cobalamin. Prodrug activation: The prodrug aurothiomalate, used for the treatment of rheumatoid arthritis, is activated by cyanide and is believed that a CN‐gold complex is the actual mediator of its beneficial effect. Cyanohydrin formation: Due to its marked nucleophilicity, cyanide is particularly reactive toward electrophiles, such as carbonyl groups of aldehydes and ketones, thus forming cyanohydrin adducts. PLP‐CN complex formation: The reaction of cyanide with the aldehydic moiety of PLP has been shown to interfere PLP‐dependent enzymes. NAD⁺‐CN complex formation: The reaction of cyanide with the C‐4 of NAD⁺ has been shown to interfere with the activity of some dehydrogenases. Cyanylation: The reaction of cyanide with protein disulfide bridge leads to cyanylation, an emerging post‐translational modification suggested to be involved in the regulation of pivotal cellular pathways (*observation made in Arabidopsis thaliana). Carbamylation: HCNO has been shown to be involved in cellular aging by carbamylating target proteins, a post‐translational modification leading protein misfunctioning. De‐glutathionylation: Cyanide reacts with the cysteine‐glutathione disulfide of glutathionylated protein, thus displacing glutathione and restoring the protein thiol.

Fig. 5

Elimination pathways of cyanide. CN^‐ is manly catabolized into thiocyanate (SCN^‐) by the sulfurtransferase enzymes and excreted as such in the urine. SCN^‐ exists in a metabolic equilibrium with the cyanide pool. The possible metabolic fates of the cyanide pool include i) interaction with cobalamin, thus forming cyanocobalamin; ii) conversion to formate, which, in turn, can be exhaled as CO₂, excreted in the urine, or take part to the one‐carbon metabolism; iii) conversion to HCNO and consequent exhalation as CO₂; iv) exhalation of free CN^‐. Minor pathways involve v) a reaction with cystine and consequent generation of 2‐iminothiazolidine‐4‐carboxylic acid (ITCA), in tautomeric equilibrium with ATCA, and vi) various additional reactions with reactive sulfur species such as persulfides, polysulfides, and nitrosopersulfides.

Fig. 6

Role of low and high cyanide concentration in the regulation of mammalian cell function in health and disease. At low (nanomolar to low micromolar) concentrations, cyanide exerts beneficial and regulatory roles, for example, stimulation of cellular bioenergetics, induction of a cytoprotective phenotype. At medium concentrations, the effect of cyanide includes the regulation of NMDA receptors and the modulation of intracellular calcium handling, which may either exert physiological or pathophysiological roles, depending on the circumstance. At high (high micromolar to millimolar) concentrations, cyanide induces calcium overload and mitochondrial dysfunction, culminating in apoptotic, or necrotic cell death.